Nucleic acid probes are often used to identify the presence of specific target sequences in genomic or amplified DNA. The annealing or melting temperature of the probe to the target is affected by the length of the complementary region shared by the probe and the target, and by the existence of any mismatches between the otherwise complementary base pairs. This can be used to detect the presence of variants, for example SNPs or multiple repeats. A probe can be designed to have a first melting temperature (Tm) to a wild type sequence, and the annealing of the probe to the target monitored, for example through development of fluorescence on annealing. If the Tm is different to the expected value, then the target sequence includes a variant.
International patent application WO2012/093262 describes methods for detecting and analysing single nucleotide polymorphisms (SNPs) using oligonucleotide probes which hybridise to variant alleles with a lower Tm than that with which they hybridise to wild type alleles. The methods use the polymerase chain reaction (PCR) to amplify a fragment of the genome including the target sequence at a temperature between the first and second Tms. If the target sequence is wild type, then the probe remains bound to the target, and prevents amplification; if the target sequence is a variant, then the probe is not bound to the target, and amplification takes place. In this way, the presence of a variant may be established, and the variant allele selectively enriched in a sample.
International patent application WO2013/041853 describes probes for detecting polymorphisms including SNPs and short tandem repeats (STRs). The probes include first and second regions joined by a linker nucleic acid sequence, such that the first and second regions have independent Tms. The probe sequences may be designed so as to have varying Tm depending on whether a variant or wild type allele is present at a first target region and a second target region. The use of this linker probe allows a single oligonucleotide probe to be used to detect variants in a longer sequence than would otherwise be possible with conventional probes.
However, not all variants are clinically important. In particular, although some mutations may be associated with phenotypic variation (for example, susceptibility to a particular drug), others may be phenotypically neutral or even silent. Silent mutations in particular are those where a mutation in the nucleotide sequence does not give rise to a corresponding mutation in the encoded polypeptide sequence. This is typically the case with mutations in the third base of a particular codon.
The following table, taken from http://en.wikipedia.org/w/index.php?title=Genetic_code&oldid=567109686, shows the genetic code and illustrates the degeneracy of the code and shows which mutations may be phenotypically silent. For example, a mutation from UUU to UUC will still code for phenylalanine, so will have no effect on the expressed protein.